The present invention, in some embodiments thereof, relates to proteasome inhibitors and uses thereof.
The proteasome is the major proteolytic complex, responsible, in eukaryotic cells, for the degradation of a multitude of cellular proteins. This multi-protein complex, present in both the cytoplasm and the nucleus, catalyzes the ATP-dependent proteolysis of short-lived regulatory proteins, as well as the rapid elimination of damaged and abnormal proteins. The 26S proteasome is a large complex of ˜2.5 MDa. Based on biochemical analyses, this complex can be dissociated into two functionally distinct subcomplexes, the 20S core particle (CP) which is the proteolytic component, and the 19S regulatory particle (RP), that is responsible for recognizing, unfolding, and translocating polyubiquitinated substrates into the 20S CP, where they are degraded.
The 20S CP is a 670 kDa barrel-shaped protein complex made up of four stacked, seven-membered rings (4×7 subunits), two outer a rings and two inner β rings (α1-7β1-7β1-7α1-7). The two matching a rings are situated in the outer rims of the barrel, facing the 19S regulatory complex. The proteolytic active sites are located on the two identical β-rings, which are positioned in the center of the 20S complex. In eukaryotes, the catalytic activities of the proteasomes are confined to only three of the β-subunits. Although proteasomes can hydrolyze the amide bonds between most amino acids, proteolytic activities measured using fluorogenic substrates define three distinct (although not conclusive) cleavage preferences [5]: β2 possesses tryptic activity (i.e., cleaving after basic residues); β5 displays chymotryptic activity (i.e., cleaving after hydrophobic residues); and β1 has “caspase-like” or “post-acidic” activity. In all three active β-subunits, proteolytic activity is associated with their N-terminal threonine residue, which acts as a nucleophile in peptide-bond hydrolysis.
The use of proteasome inhibitors as drug candidates emerged from the observation that at specific concentrations, they can induce apoptosis in certain leukemia- and lymphoma-derived cells without similarly affecting their non-transformed counterparts. Further development and clinical trials led to the approval of the modified boronic dipeptide Pyz-Phe-boroLeu, known as Bortezomib as a drug for the treatment of multiple myeloma. Most synthetic proteasome inhibitors are short peptides that mimic protein substrates. Typically, the pharmacophore that reacts with and inhibits the threonine residue in the 20S proteasome's active site is bound to the carboxyl residue of the peptide. Some of the typical synthetic inhibitors are peptide aldehydes, peptide vinyl sulfones, peptide boronates, and peptide epoxyketones. Most notable among the natural, bacterially derived non-peptide inhibitors is claso-lactacystin-β-lactone (Omuralide). Related drugs such as Salinosporamide A (NPI-0052) and Carfilzomib (PR-171) are currently in advanced clinical trials. However, despite the extensive efforts invested in proteasome inhibitor development, there is a growing need for novel inhibitory molecules, due to the emergence of drug-resistant cells and the variable effects of existing inhibitors on different cells.
Most of the current assays for proteasome inhibition are based on cell-free assays, which require purification of 26S or 20S proteasomes from different sources. Such assays may, in principle, be adapted to high-throughput screens, yet they may fail to predict the inhibitory activity in live cells. To overcome this problem, cell-based screens have been incorporated into the drug discovery process. For example, a modified “classical” method for measurement of the chymotrypsin-like, trypsin-like, or caspase-like proteasome activities in cultured cells [Moravec R A et al., 2009, Anal Biochem 387: 294-302] is currently available from Promega Corporation. A number of fluorescent reporter molecules have been also usefully employed to monitor the activity of the proteasome. Dantuma et al constructed a fusion of GFP to Ubiquitin (Ubi[G76V]-GFP) using a standard peptide bond at the N-terminus [Nat Biotechnol 18: 538-543, 2000], Another proteasome sensor construct, which is a GFP fusion to an artificial peptide, CL1, identified in yeast has been designed by Bence et al (Science 292: 1552-1555, 2001). The Andreatta group and BD Biosciences Clontech has introduced a sensor cell line expressing a GFP fusion protein with a fragment of the mouse ornithine decarboxylase (MODC), which is degraded by the proteasome without the requirement for ubiquitination [Andreatta et al, 2001, Biotechniques 30: 656-660]. An additional reporter cell line, based on the stable expression of a p27kip1-GFP fusion was recently employed for the discovery of a novel proteasome inhibitor, argyrin A [Nickeleit I et al., 2008, Cancer Cell 14: 23-35]. The common feature of most of these GFP-fused reporters is that they are based on proteins rapidly degraded by the proteasome under normal conditions, leading to very low fluorescence of the cells, while following inhibition of proteasome activity, the overall fluorescent signal of the cells rapidly increases as a result of accumulation of the reporter proteins.